Mixed plug-flow loop reactor (MPFLR) has been widely adopted by the US dairy farms to convert cattle manure to biogas. However, the microbiome in MPFLR digesters remains unexplored. In this study, the microbiome in a MPFLR digester operated on a mega-dairy farm was examined thrice over a 2 month period. Within 23 days of retention time, 55-70% of total manure solid was digested. Except for a few minor volatile fatty acids (VFAs), total VFA concentration and pH remained similar along the course of the digester and over time. Metagenomic analysis showed that although with some temporal variations, the bacterial community was rather stable spatially in the digester. The methanogenic community was also stable both spatially and temporally in the digester. Among methanogens, genus Methanosaeta dominated in the digester. Quantitative polymerase chain reaction (qPCR) analysis and metagenomic analysis yielded different relative abundance of individual genera of methanogens, especially for Methanobacterium, which was predominant based on qPCR analysis but undetectable by metagenomics. Collectively, the results showed that only small microbial and chemical gradients existed within the digester, and the digestion process occurred similarly throughout the MPFLR digester. The findings of this study may help improve the operation and design of this type of manure digesters.